Project Summary/Abstract Proper functions of the brain rely on specific neuron connections and their assembly into functional circuit. However, many detailed mechanisms of the dynamic process of circuit assembly are unclear due to difficulty to visualize it in live brains. Employing an explant system based live imaging, here I propose to investigate the cell biology underpinnings of neural circuit assembly using the Drosophila olfactory system. In the Drosophila olfactory circuit, 50 classes of olfactory receptor neurons (ORNs) send their axons to specific glomeruli in the antennal lobe (AL), where they make one-to-one connections with corresponding second-order olfactory projection neurons (PNs). This provides an excellent system to study how neurons form specific connections during circuit assembly. Recently I developed an explant system that allows me to use live imaging to reveal the dynamic process of olfactory circuit assembly. Here I propose to use this explant to: 1. image how single ORN axon targets its glomerulus; 2. explore how ORN-ORN or ORN-PN interaction regulates targeting through surgical cutting one antennal nerve; 3. characterize the mistargeting phenotype in Drosophila latrophilin mutant using live imaging (Aim 1 and part of Aim 3, K99 training phase). The K99 training phase studies mostly focus on defining the coarse targeting using two-photon microscope based long term imaging. To further pursue detailed mechanisms underlying interesting behaviors of the ORN axons, advanced imaging method allowing higher temporal and spatial resolution is required. Therefore I propose to perform high speed two color imaging using adaptive optical lattice lightsheet microscope. Combined with genetic tool for cytoskeleton labeling and manipulation, I aim to understand cell biology mechanism of crucial steps in ORN axon targeting. This study will be in collaboration with Dr. Eric Betzig at Janelia Research Campus. We have already got promising results from pilot experiments. I will also pursue potential Latrophilin-Ten-a mediated axon-axon or axon-dendrite repulsion using live imaging (Aim 2 and part of Aim 3, R00 training phase). In general the R00 training phase studies will deal with more complex situation in both imaging strategy and biological context. My mentoring team consists of Dr. Liqun Luo (mentor), a world-renowned neuroscientist, and Drs. Kang Shen (co-mentor), an experienced cell biologist and developmental neurobiologist and Eric Betzig (collaborator), Nobel laureate and world-renowned microscopist, as well as Dr. Michael Lin (consultant), an expert on developing genetic imaging tools. With their guidance, I believe that I will gain extensive training in my transition to be an independent PI.